HISTOLOGY FULL-TEXT

William A Beresford MA, D Phil ©
Professor of Anatomy
Anatomy Department, West Virginia University, Morgantown, USA

Chapter 1 HISTOLOGY: METHOD AND MICROSCOPY

A NATURE OF HISTOLOGY

Medical histology applies microscopy to the human body, seeking to discover the nature of its smaller structures, how they relate to each other, and what they do. Thinking in histology runs along these lines. How does one prepare living and dead tissues for microscopy to best keep their images faithful to their true nature? What kinds of microscopy can be applied? How does one analyse and describe the images yielded at different orders of magnification by the various microscopes? Does the microscopic appearance of the tissue or cell suggest something of how it works, its chemical nature, and what may go wrong in disease? What experiments can one do to test ideas on how structure relates to function?

The answers comprise a large body of knowledge graced in several ways. First, histology is colourful. Secondly, almost everything seen is actually there; which is not to say that what is not seen is absent. Third, one handles and views actual slides - the source material for most of histology, not just someone else's selected images. Fourth, the structures can be interpreted as parts in developmental and functional sequences, and be fitted together by satisfying accounts, for example, of how cells defend the body. So much is now known of the roles of cells and structures that histology is both descriptive microanatomy, and an introduction to function for the whole body. Powerpoint
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B PREPARATION OF THE MATERIAL

1 A major distinction can be drawn between dead and living preparations
            Dead                          Living
(a)Section - a thin slice of           Such preparations may be out of the
   tissue or organ - on a glass slide  body in a tissue culture system, or
   or metal grid.                      living within the body but in an 
(b)Smear on a glass slide -            observable situation, e.g., a  
   suitable for suspensions, e.g.,     transparent chamber inserted into 
   blood, urine, mucus, cyst fluid,    the ear or skin. The  first need is 
   bone marrow, etc.                   to keep the preparation alive. This  
(c)Spread sheet of tissue              seriously limits the facilities for
   stretched thin, e.g., areolar       observation. For example, staining
   connective tissue.                  is usually impracticable. Thus,   
(d)Teased apart fibrous                phase-contrast or interference-contrast
   elements, e.g., muscle.             microscopy has to be used in order
                                       to overcome the poor contrast
                                       between natural structures.

2 Steps needed to make and study a histological section

  1. Fixation to prevent post-mortem decomposition, preserve structure, and intensify subsequent staining.
  2. (a) Steps involved in imbedding the tissue in a block of wax or plastic, or (b) freezing of the material to a firm mass, for
  3. cutting into thin sections on a microtome; 1-l50 microns (µm) thick for light microscopy (LM); 30-60 nanometres (nm) for electron microscopy (EM).
  4. Units: based on the metre (m): micron/micrometre (µm) = 10-6m; nanometre (nm)/ millimicron (mµ) = l0-9m; Ångström (Å) = l0-10m; l0Å=1nm.
  5. Mounting of the section on a glass slide or metal grid. Staining of the section with one or more reagents, e.g., solutions of metallic salts, in one or more stages.
  6. For light microscopy, the removal of surplus stain and water, and steps involved in holding a thin glass coverslip to the section with a mounting medium having a refractive index close to that of glass.
  7. Observation and recording by means of the microscope, and notes, photomicrography, projection drawing, labelled sketches, counting and reconstructions, digital and videorecording. A drawback to using our eyes as part of the observing instrument is that the visual system does not record accurately. Memory is unreliable.

C MICROSCOPY

1 Microscopy in general The main distinction is between light microscopy and electron microscopy. The usual light microscope uses a visible light source with a system of condenser lenses to send the light through the object to be examined. The image of this object is then magnified by two sets of lenses, the objective and the eyepiece. Total magnification is then the product of these two lens systems, e.g., 40 X 10 = 400. The resolution or resolving power - how close two structures can be and still be seen as separate - is a measure of the detail that can be seen, and for the light microscope is about 0.25 µm. This limit to resolution is determined mostly by the wavelength of the light; and, however powerful the lens, 0.25 µm cannot be improved upon.

The only way to improve resolving power is to reduce substantially the wavelength of the light. This is achieved by the electromagnetic beam of the electron microscope. The beam is focused through the object suspended on its metal grid, and is magnified before striking a fluorescent screen to be transformed into a visible image (Chapter 30.K). [Caution! the link takes you there , but 'back' brings you only to the start of the last Chapter that you linked to.]
The resolutions so far achieved in biology with transmission electron microscopy (TEM/EM) are of the order of 1 nm at a magnification of X 2 000 000. The other forms of microscopy - phase-contrast, interference, fluorescence, confocal scanning, atomic-force (and X-ray diffraction) - will be discussed in Chapter 30. in relation to the problems for which they are suited.

2 Microscopy for the student (may not apply in toto to the reader's use)

  1. The usual class microscope has eyepieces/oculars magnifying X 10, and an objective nosepiece carrying X 4, X 10, X 44, and X 95 (oil immersion) lenses. Normally the three lower-power lenses are kept mounted on the nosepiece, whilst the oil immersion objective may be mounted or kept separately.
  2. Every time it is used, the microscope should be set up to the best optical advantage. How to do this is described briefly below.
  3. Keep in mind the limit to resolution. In practical terms, make special note of those structures that need an oil immersion lens to be seen or are visible only in electron microscopy.
  4. The section has some thickness, so that the fine-focusing adjustment should be used continually during observation to bring out fine detail, e.g., cilia on cells. Essentially, though, we are getting a two-dimensional picture from an originally three-dimensional piece of material. For what the structure looked like in the third dimension, the student can try to reconstruct mentally what is going on in the missing dimension, and look up views of the structure in scanning electron microscopy.
  5. Artefacts (appearances not due to the original nature of the material as obtained from the body) can arise at all stages in the treatment of the section. Gross examples arise from: (l) clumsy excision from the body; (2) poor or inappropriate fixation; (3) shrinkage and, worse, uneven shrinkage, leading to artificial spaces and distorted relations; (4) cutting scores from a bad microtome knife; (5) the section not flat on the slide; (6) water, dirt or bubbles on or in the section; (7) dirt on the microscope lenses; (8) patchy or faded staining; unbalanced staining when more than one stain has been applied; (9) precipitate from fixative or stain; (l0) tears and folds in the section.
  6. Setting up the microscope
    (a) Ask for and read the instruction booklet for the 'scope.
    (b) Familarize yourself with the parts and controls of the microscope, in particular with the: (your 'scope may not have all these)
    ... (i) light source and switch,
    ... (ii) two-sided mirror, if present,
    ... (iii) iris diaphragm lever on the condenser assembly,
    ... (iv) condenser focusing knob,
    ... (v) stage slide clips and controls for a mechanically movable stage,
    ... (vi) objective selection on the three- or four-way objective nose-piece
    ... (vii) coarse focusing adjustment,
    ... (viii) fine focusing adjustment,
    ... (ix) pointer in the eyepiece lens,
    ... (x) any control for eyepiece focusing.

(c) Before plugging in the microscope, check to feel how the switch and rheostat work. Plug in, switch on, and adjust the rheostat up one third of its range to start.
(d) Otherwise, use artificial light provided by an electric bulb behind a ground-glass screen to furnish a constant and reliable source. Light intensity can be increased by bringing the lamp nearer to the mirror, if the lamp is not built-in.
(e) If the condenser in use (nearly always), use the plane side of the mirror, if the lamp is not built-in.
(f) Raise the condenser to very near the underside of the stage, and open the iris diaphragm.
(g) Place a clean, stained slide on the stage and using the coarse and fine focusing controls bring it into focus with X l0 objective.
(h) With the condenser racking knob focus the light source on the specimen. This has happened when the specimen itself is in focus and some aspects of the light source is also seen sharply defined, e.g., the bulb filament or scratches on the frosted glass screen. If this feature of the light source is obtrusive, now place the condenser very slightly out of focus. Do not lower the condenser way out of focus as a means to reduce the light intensity.
(i) The iris diaphragm should now be closed just to the point where glare is eliminated. Further closure will make the field too dark and reduce resolving power.
(j) The microscope is now set up for use, but the requirements change for each objective. Higher power objectives require more light thus the iris will need to be opened and perhaps the lamp brought nearer to the mirror and the condenser refocused.
(k) Note that the objective lenses are of different lengths, and they are not always parfocal. Be careful when switching in a higher power lens that it does not hit the slide because of its greater length. Clean the lenses only with lens paper.
(l) If the X 44 objective will not focus to a clear image, check first that the slide is not upside down on the stage.

(m) Use of the 'oil immersion' lens:
... (i) Select field of interest with the high dry lens (X40); centre precisely the cell or object in the microscopic field; if X 95 lens is already mounted go to (v).
... (ii) Raise the objective lens assembly and remove the low power (X4) lens.
... (iii) Place it in the container to be found on the door of the microscope cabinet from which you have taken the oil immersion (X 95) lens.
... (iv) Screw the oil lens into the now vacant place on the objective nosepiece.
... (v) Place carefully one drop of immersion oil from the small bottle issued on the area of the slide to be studied.
... (vi) Switch round the objective nosepiece to bring the oil immersion lens into play.
... (vii) Very carefully lower the objective assembly with the coarse focusing, until the tip of the oil lens touches the drop of oil. This operation must be controlled by observing the descending lens from the side. Do not yet look down through the eyepiece. Once the lens has touched the oil raise it slightly, but not so far that the drop breaks away.
... (viii) Look in the eyepiece and focus down with the fine focusing control very slowly and gently until the specimen comes into focus. If you seem to have gone down a very long way without a clear image, again check from the side that you have not overshot and the lens is not nearly on the glass of the coverslip. If this has happened raise the lens slowly, while looking for a focused image.
... (ix) The oil objective lens needs much light so that the iris diaphragm may have to be opened.
... (x) As soon as you have finished using the oil lens, raise (remove) and clean it. (Replace X 4 lens on the nosepiece and the oil lens in its box.) Clean the slide of oil with lens or tissue paper. Do not allow oil to get on to the other, dry, lenses.

(n) Other controls the class microscope may have include eyepiece focusing, filter-holders, centring screws for the condenser, and a rheostat, lens and light-stop for the light source. Ask for instructions in their use and for help with any mechanical problem.

(o) Take care of the microscope, carry it only by its arm, protect it from dust by keeping it locked in its case, and do not stand it or boxes of slides near the edge of the bench. If lens paper alone is insufficient to clean a lens, use no solvents but consult a demonstrator. On no account exchange the lenses of your microscope for those of any other microscope.

Spectacle-wearers need not use their glasses in microscopy; if they do, they should beware of damaging their glasses, while trying to compensate for the narrowed field of view.

3 Differences between light and electron microscopy
l Chapters 2 and 3 deal with microscopic details of cells - cytology, for which EM is better suited than LM. Table l gives some differences between the two approaches. The detailed morphology revealed by EM may be called fine or submicroscopic structure/ultrastructure.
2 The direct comparison of LM and EM images of a structure requires that the magnifications be of the same order. Noting the magnification, on the 'scope or in the figure legend, allows one to adjust one's expectations of what may be seen, and should always be done.
3 A growing practice in histology and pathology is to fix and prepare the tissue by EM standards, imbed in plastic and cut semi-thin (l µm) sections for staining by modified LM methods. LM then reveals good cellular detail and fewer artefacts.
4 Two other techniques yield anatomical images - fibre-optic endoscopy and scanning EM, and are being digested by the anatomical texts. Endoscopy from its low magnification is marginal to histology, but related in that endoscopy is used to obtain biopsy specimens for histopathology.
SEM strengthens one's conception of microscopic structures, e.g., cilia, renal podocytes, bone under resorption, and effectively counters the unavoidable impression of structures existing only in two dimensions. (From hereon, EM is standard transmission electron microscopy.)

Table 2. Some differences between light and electron microscopy.

Light microscopy                        Electron microscopy
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Image is presented directly to the     Image is in shades of green on
eye. Image keeps the colours given     the screen; photographically,
the specimen by staining.              only in black and white.                                       

Modest magnification to X 1500;        High magnification, up to X 2,000,000
but a wider field of view and easier   thus the range of magnification
orientation                                        is greater
                           

Resolving power to 0.25 µm.            Resolving power to 1 nm (0.001µm.)

Frozen sections can yield an image     Processing of tissue takes a day at
within 20 minutes.                     least.

Crude techniques of preparation        High resolution and magnification
introduce many artefacts.              demand good fixation (e.g. by
(Histochemical methods are better.)    vascular perfusion), cleanliness
                                       and careful cutting, adding up to
                                       fewer artefacts.

Section thickness (1-30 µm) gives      Very thin sections provide no
a little depth for focus for           depth of focus, but 3-D information
appreciation of the third dimension.   can be had from: (a) thicker sections
Serial sections can be cut, viewed     by high-voltage EM; (b) shadowed
and used to build a composite image    replicas of fractured surfaces; (c)
or representation.                     scanning electron microscopy (SEM).

Most materials and structures cannot   Heavy metal staining gives a more
be stained and viewed at the same      comprehensive picture of membranes,
time; stains are used selectively to   granules, filaments, crystals, etc.;
give a partial picture, e.g. a stain   but this view is incomplete and even
for mucus counterstained to show       visible bodies can be improved by
cell nuclei.                           varying the technique.

Specimen can be large and              Specimen is in vacuo. Its small size
even alive.                            creates more problems with sampling
                                       and orientation.                 
 
Light microscopy                       Electron microscopy

William A Beresford, Anatomy Department, School of Medicine, West Virginia University, Morgantown, WV 26506-9128, USA - - e-mail: -- wberesfo@wvu.edu -- wberesfo@hotmail.com -- beresfo@wvnvm.wvnet.edu -- fax: 304-293-8159